High-Throughput Engineering of a Light-Activatable Ca2+ Channel

The current platform for optical control of calcium (Ca²⁺) entry into mammalian cells, including those built upon microbial channelrhodopsin-2 (ChR2) or STIM1-based photoactivatable Ca²⁺ release-activated Ca²⁺ (Opto-CRAC) channel, either lacks Ca²⁺ selectivity or requires the presence of endogenous ORAI Ca²⁺ channel for functionality. In this chapter, we describe the engineering of a bona fide blue light-operated Ca²⁺ (LOCa) channel. We present detailed protocols for the generation of LOCa by incorporating blue light photosensory LOV2 domain into the highly Ca²⁺ specific ORAI1 channel via a high-throughput screening method. The optimized single component LOCa enables reversible Ca²⁺ influx without the need for exogenous cofactors. Our method can also be readily extended for optogenetic engineering of other types of ion channels.